ptdTomato

ptdTomato

ptdTomato载体基本信息

ptdTomato载体质粒图谱和多克隆位点信息

ptdTomato载体描述

ptdTomato encodes tdTomato, a member of the family of fruit fluorescent proteins (1) derived from the Discosoma sp. red fluorescent protein, DsRed (2). Because the Tomato protein has a tendency to dimerize, the vector was desighed with two copies of the Tomato coding region liked together to allow intramolecular dimerization. As a result, each tdTomato RNA transcript encodes a tandem dimer of the Tomato protein (excitation and emission maxima equal 554nm and 581nm, respectively). In ptdTomato, the tdTomato coding sequence (2) is flanked by restriction sites (5' MCS and 3' MCS) that make it easy to excise the gene for use in other cloning applications. In E. coli, tdTomato is expressed from the lac promoter (Plac) as a fusion with several amino acids, including the first five amino acids of the LacZ protein. Note, however, that if the tdTomato coding sequence is excised using a restriction site in the 5' MCS, the protein will no longer be expressed as a fusion (as it is when expressed from the lac promoter).A Kozak consensus sequence is located immediately upstream of the tdTomato coding sequence to enhance translational efficiency in eukaryotic cells (3). In the tdTomato vector, the entire tdTomato expression cassette is supported by a pUC19 backbone, which contains a high copy-number origin of replication and an ampicillin resistance gene for propagation and selection in E. coli.

The ptdTomato vector is primarily intended to serve as a source of tdTomato cDNA. The flanking MCS regions make it possible to excise the tdTomato coding sequence and insert it into other vector systems. The vector can also be used to express tdTomato in bacteria.

For Western analysis, either the Living Colors DsRed Polyclonal Antibody (Cat. No. 632496) or DsRed Monoclonal Antibody (Cat. Nos. 632392 and 632393) can be used to detect the tdTomato protein.

Propagation in E. Coli

 Recommended host strain: DH5α

 Selectable marker: plasmid confers resistance to ampicillin (50 μg/ml) in E. coli hosts.

 E. coli replication origin: pUC

 Copy number: high

 Plasmid incompatibility group: pMB1/ColE1

 Excitation and emission maxima of tdTomato

 Excitation maximum = 554 nm

 Emission maximum = 581 nm

ptdTomato载体序列

ORIGIN

    1 AGCGCCCAAT ACGCAAACCG CCTCTCCCCG CGCGTTGGCC GATTCATTAA TGCAGCTGGC

   61 ACGACAGGTT TCCCGACTGG AAAGCGGGCA GTGAGCGCAA CGCAATTAAT GTGAGTTAGC

  121 TCACTCATTA GGCACCCCAG GCTTTACACT TTATGCTTCC GGCTCGTATG TTGTGTGGAA

  181 TTGTGAGCGG ATAACAATTT CACACAGGAA ACAGCTATGA CCATGATTAC GCCAAGCTTG

  241 CATGCCTGCA GGTCGACTCT AGAGGATCCC CGGGTACCGG TCGCCACCAT GGTGAGCAAG

  301 GGCGAGGAGG TCATCAAAGA GTTCATGCGC TTCAAGGTGC GCATGGAGGG CTCCATGAAC

  361 GGCCACGAGT TCGAGATCGA GGGCGAGGGC GAGGGCCGCC CCTACGAGGG CACCCAGACC

  421 GCCAAGCTGA AGGTGACCAA GGGCGGCCCC CTGCCCTTCG CCTGGGACAT CCTGTCCCCC

  481 CAGTTCATGT ACGGCTCCAA GGCGTACGTG AAGCACCCCG CCGACATCCC CGATTACAAG

  541 AAGCTGTCCT TCCCCGAGGG CTTCAAGTGG GAGCGCGTGA TGAACTTCGA GGACGGCGGT

  601 CTGGTGACCG TGACCCAGGA CTCCTCCCTG CAGGACGGCA CGCTGATCTA CAAGGTGAAG

  661 ATGCGCGGCA CCAACTTCCC CCCCGACGGC CCCGTAATGC AGAAGAAGAC CATGGGCTGG

  721 GAGGCCTCCA CCGAGCGCCT GTACCCCCGC GACGGCGTGC TGAAGGGCGA GATCCACCAG

  781 GCCCTGAAGC TGAAGGACGG CGGCCACTAC CTGGTGGAGT TCAAGACCAT CTACATGGCC

  841 AAGAAGCCCG TGCAACTGCC CGGCTACTAC TACGTGGACA CCAAGCTGGA CATCACCTCC

  901 CACAACGAGG ACTACACCAT CGTGGAACAG TACGAGCGCT CCGAGGGCCG CCACCACCTG

  961 TTCCTGGGGC ATGGCACCGG CAGCACCGGC AGCGGCAGCT CCGGCACCGC CTCCTCCGAG

 1021 GACAACAACA TGGCCGTCAT CAAAGAGTTC ATGCGCTTCA AGGTGCGCAT GGAGGGCTCC

 1081 ATGAACGGCC ACGAGTTCGA GATCGAGGGC GAGGGCGAGG GCCGCCCCTA CGAGGGCACC

 1141 CAGACCGCCA AGCTGAAGGT GACCAAGGGC GGCCCCCTGC CCTTCGCCTG GGACATCCTG

 1201 TCCCCCCAGT TCATGTACGG CTCCAAGGCG TACGTGAAGC ACCCCGCCGA CATCCCCGAT

 1261 TACAAGAAGC TGTCCTTCCC CGAGGGCTTC AAGTGGGAGC GCGTGATGAA CTTCGAGGAC

 1321 GGCGGTCTGG TGACCGTGAC CCAGGACTCC TCCCTGCAGG ACGGCACGCT GATCTACAAG

 1381 GTGAAGATGC GCGGCACCAA CTTCCCCCCC GACGGCCCCG TAATGCAGAA GAAGACCATG

 1441 GGCTGGGAGG CCTCCACCGA GCGCCTGTAC CCCCGCGACG GCGTGCTGAA GGGCGAGATC

 1501 CACCAGGCCC TGAAGCTGAA GGACGGCGGC CACTACCTGG TGGAGTTCAA GACCATCTAC

 1561 ATGGCCAAGA AGCCCGTGCA ACTGCCCGGC TACTACTACG TGGACACCAA GCTGGACATC

 1621 ACCTCCCACA ACGAGGACTA CACCATCGTG GAACAGTACG AGCGCTCCGA GGGCCGCCAC

 1681 CACCTGTTCC TGTACGGCAT GGACGAGCTG TACAAGTAGG CGGCCGCGAC TCTAGAATTC

 1741 CAACTGAGCG CCGGTCGCTA CCATTACCAA CTTGTCTGGT GTCAAAAATA ATAGGCCTAC

 1801 TAGTCGGCCG TACGGGCCCT TTCGTCTCGC GCGTTTCGGT GATGACGGTG AAAACCTCTG

 1861 ACACATGCAG CTCCCGGAGA CGGTCACAGC TTGTCTGTAA GCGGATGCCG GGAGCAGACA

 1921 AGCCCGTCAG GGCGCGTCAG CGGGTGTTGG CGGGTGTCGG GGCTGGCTTA ACTATGCGGC

 1981 ATCAGAGCAG ATTGTACTGA GAGTGCACCA TATGCGGTGT GAAATACCGC ACAGATGCGT

 2041 AAGGAGAAAA TACCGCATCA GGCGGCCTTA AGGGCCTCGT GATACGCCTA TTTTTATAGG

 2101 TTAATGTCAT GATAATAATG GTTTCTTAGA CGTCAGGTGG CACTTTTCGG GGAAATGTGC

 2161 GCGGAACCCC TATTTGTTTA TTTTTCTAAA TACATTCAAA TATGTATCCG CTCATGAGAC

 2221 AATAACCCTG ATAAATGCTT CAATAATATT GAAAAAGGAA GAGTATGAGT ATTCAACATT

 2281 TCCGTGTCGC CCTTATTCCC TTTTTTGCGG CATTTTGCCT TCCTGTTTTT GCTCACCCAG

 2341 AAACGCTGGT GAAAGTAAAA GATGCTGAAG ATCAGTTGGG TGCACGAGTG GGTTACATCG

 2401 AACTGGATCT CAACAGCGGT AAGATCCTTG AGAGTTTTCG CCCCGAAGAA CGTTTTCCAA

 2461 TGATGAGCAC TTTTAAAGTT CTGCTATGTG GCGCGGTATT ATCCCGTATT GACGCCGGGC

 2521 AAGAGCAACT CGGTCGCCGC ATACACTATT CTCAGAATGA CTTGGTTGAG TACTCACCAG

 2581 TCACAGAAAA GCATCTTACG GATGGCATGA CAGTAAGAGA ATTATGCAGT GCTGCCATAA

 2641 CCATGAGTGA TAACACTGCG GCCAACTTAC TTCTGACAAC GATCGGAGGA CCGAAGGAGC

 2701 TAACCGCTTT TTTGCACAAC ATGGGGGATC ATGTAACTCG CCTTGATCGT TGGGAACCGG

 2761 AGCTGAATGA AGCCATACCA AACGACGAGC GTGACACCAC GATGCCTGTA GCAATGGCAA

 2821 CAACGTTGCG CAAACTATTA ACTGGCGAAC TACTTACTCT AGCTTCCCGG CAACAATTAA

 2881 TAGACTGGAT GGAGGCGGAT AAAGTTGCAG GACCACTTCT GCGCTCGGCC CTTCCGGCTG

 2941 GCTGGTTTAT TGCTGATAAA TCTGGAGCCG GTGAGCGTGG GTCTCGCGGT ATCATTGCAG

 3001 CACTGGGGCC AGATGGTAAG CCCTCCCGTA TCGTAGTTAT CTACACGACG GGGAGTCAGG

 3061 CAACTATGGA TGAACGAAAT AGACAGATCG CTGAGATAGG TGCCTCACTG ATTAAGCATT

 3121 GGTAACTGTC AGACCAAGTT TACTCATATA TACTTTAGAT TGATTTAAAA CTTCATTTTT

 3181 AATTTAAAAG GATCTAGGTG AAGATCCTTT TTGATAATCT CATGACCAAA ATCCCTTAAC

 3241 GTGAGTTTTC GTTCCACTGA GCGTCAGACC CCGTAGAAAA GATCAAAGGA TCTTCTTGAG

 3301 ATCCTTTTTT TCTGCGCGTA ATCTGCTGCT TGCAAACAAA AAAACCACCG CTACCAGCGG

 3361 TGGTTTGTTT GCCGGATCAA GAGCTACCAA CTCTTTTTCC GAAGGTAACT GGCTTCAGCA

 3421 GAGCGCAGAT ACCAAATACT GTTCTTCTAG TGTAGCCGTA GTTAGGCCAC CACTTCAAGA

 3481 ACTCTGTAGC ACCGCCTACA TACCTCGCTC TGCTAATCCT GTTACCAGTG GCTGCTGCCA

 3541 GTGGCGATAA GTCGTGTCTT ACCGGGTTGG ACTCAAGACG ATAGTTACCG GATAAGGCGC

 3601 AGCGGTCGGG CTGAACGGGG GGTTCGTGCA CACAGCCCAG CTTGGAGCGA ACGACCTACA

 3661 CCGAACTGAG ATACCTACAG CGTGAGCTAT GAGAAAGCGC CACGCTTCCC GAAGGGAGAA

 3721 AGGCGGACAG GTATCCGGTA AGCGGCAGGG TCGGAACAGG AGAGCGCACG AGGGAGCTTC

 3781 CAGGGGGAAA CGCCTGGTAT CTTTATAGTC CTGTCGGGTT TCGCCACCTC TGACTTGAGC

 3841 GTCGATTTTT GTGATGCTCG TCAGGGGGGC GGAGCCTATG GAAAAACGCC AGCAACGCGG

 3901 CCTTTTTACG GTTCCTGGCC TTTTGCTGGC CTTTTGCTCA CATGTTCTTT CCTGCGTTAT

 3961 CCCCTGATTC TGTGGATAAC CGTATTACCG CCTTTGAGTG AGCTGATACC GCTCGCCGCA

 4021 GCCGAACGAC CGAGCGCAGC GAGTCAGTGA GCGAGGAAGC GGAAG

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